The overall aims of this research are to understand the molecular mechanism by which actomyosin motility systems convert chemical energy into mechanical work, and to obtain a precise correlation between the mechanical, biochemical and structural events at the molecular level. Novel methods will be applied to isolated muscle myosin and unconventional myosin molecular motors to probe the relations between biochemical reactions of the contractile proteins, the elementary mechanical steps of the cross-bridge cycle and the corresponding structural motions. Bifunctional or bi-arsenical fluorescent probe molecules will be stably bound with known orientation to the motor domains and to light chain subunits in the myosin heads. The spatial orientation and translational position of these components will be monitored at high time resolution by novel single-molecule fluorescence polarization, total internal reflection (polTIRF) microscopy to determine the dynamics of specific protein structural changes. Increased time resolution of the rotational and translational motions will enable events during molecular steps to be determined. Nanometer resolution tracking of the molecular position in the x, y and z directions, using a new "Parallax View" technique will disclose mechanisms of molecular stepping and choice of path along the cytoskeletal filament. An infrared optical trap, with high-speed feedback to clamp the actin in place, will be combined with single-molecule polTIRF microscopy to directly evaluate the influence of mechanical stress and strain on stepping rates and protein orientation changes that relate to chemo-mechanical transduction. The energetics and probabilistic nature of stepping target selection will be determined from the orientation and force dependence of the distributions of head angle, biochemical state and step size. The experiments will be carried out on conventional myosin isolated from muscle tissue and on non-muscle myosins isolated from neural tissue and recombinant expression systems. Results from this project should significantly advance knowledge of cell motility processes and thus bring a greater understanding of both normal and pathological states of striated muscle, neuronal development and other types of cell motility. PUBLIC HEALTH RELEVANCE: Myosin molecular motors are involved, altered or compromised in many diseases, such as muscle weakness and fatigue, hypertrophic cardiomyopathy, hypertension, cell division, chemotaxis, neoplastic invasiveness and metastasis, and in many neurological diseases, such as specific forms of developmental deficits and congenital deafness and blindness. Fundamental research on these proteins thus will provide the basic understanding to enable generation of new diagnostic and therapeutic measures.